Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

Wang SC, Hsu HJ, Lin GW, Wang TF, Chang CC, Lin MD - Sci Rep (2015)

Bottom Line:
We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

ABSTRACTFormation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

f1: ApVas1 was not colocalised with Oskar (Osk) to the posterior germ plasm of Drosophila oocyte.(A) Schematic alignment of Drosophila Vasa (DmVas) and the pea aphid Vasa (ApVas1). Open boxes show the green fluorescent protein (GFP) tag, the DEAD-like helicases superfamily (DEXDc), and helicase superfamily C-terminal (HELICc) domains. Sequence identity and similarity are highlighted beneath the domain boxes. Location of the Osk interacting motif (OIM) of DmVas: amino acids 163–319. (B–D) Posterior localisation of GFP-DmVas in the oocyte of Stage-9–10 egg chambers. (E,F) Expression of GFP-ApVas1 in the late Stage-9 and Stage-10 egg chambers. Posterior localisation of GFP-ApVas1 was not identified. (B’–F’) Magnification of the insets shown in (B–F). (B”–F”) Posterior localisation of Osk. (B”’–F”’) Merged images. The egg chambers were double stained using antibodies against GFP (green) and Osk (red). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.

Mentions:
Alignment of DmVas and ApVas1, which are germline markers in Drosophila and the pea aphid, respectively, displays highly conserved features in their helicase core domains DEXDc and HELICc21 (Fig. 1A; Supplementary Fig. S1A). To understand the extent of similarity in the functions of ApVas1 and DmVas, we specifically expressed green fluorescent protein (GFP)-ApVas1 in the Drosophila female germline by using a maternal tubulin 67c promoter26. GFP-DmVas expression was driven by the same promoter and served as a positive control. As expected, the posterior localisation of DmVas to the germ plasm was observed from mid-stage 9 of oogenesis827, right after Osk could first be detected in the posterior pole of the oocytes282930 (Fig. 1B–B”’). Colocalisation of DmVas and Osk to the germ plasm became even more prominent in the egg chambers at late stage 9 (Fig. 1C–C”’) and stage 10 (Fig. 1D–D”’). However, during the same period of oogenesis, ApVas1 was not colocalised with Osk in the posterior germ plasm (Fig. 1E–E”’”’). Instead, we could only visualise a weak expression of ApVas1 in the lateral cortex of the oocytes (Fig. 1E,F).

f1: ApVas1 was not colocalised with Oskar (Osk) to the posterior germ plasm of Drosophila oocyte.(A) Schematic alignment of Drosophila Vasa (DmVas) and the pea aphid Vasa (ApVas1). Open boxes show the green fluorescent protein (GFP) tag, the DEAD-like helicases superfamily (DEXDc), and helicase superfamily C-terminal (HELICc) domains. Sequence identity and similarity are highlighted beneath the domain boxes. Location of the Osk interacting motif (OIM) of DmVas: amino acids 163–319. (B–D) Posterior localisation of GFP-DmVas in the oocyte of Stage-9–10 egg chambers. (E,F) Expression of GFP-ApVas1 in the late Stage-9 and Stage-10 egg chambers. Posterior localisation of GFP-ApVas1 was not identified. (B’–F’) Magnification of the insets shown in (B–F). (B”–F”) Posterior localisation of Osk. (B”’–F”’) Merged images. The egg chambers were double stained using antibodies against GFP (green) and Osk (red). In all panels, anterior is to the left and posterior is to the right. Scale bars, 25 μm.

Mentions:
Alignment of DmVas and ApVas1, which are germline markers in Drosophila and the pea aphid, respectively, displays highly conserved features in their helicase core domains DEXDc and HELICc21 (Fig. 1A; Supplementary Fig. S1A). To understand the extent of similarity in the functions of ApVas1 and DmVas, we specifically expressed green fluorescent protein (GFP)-ApVas1 in the Drosophila female germline by using a maternal tubulin 67c promoter26. GFP-DmVas expression was driven by the same promoter and served as a positive control. As expected, the posterior localisation of DmVas to the germ plasm was observed from mid-stage 9 of oogenesis827, right after Osk could first be detected in the posterior pole of the oocytes282930 (Fig. 1B–B”’). Colocalisation of DmVas and Osk to the germ plasm became even more prominent in the egg chambers at late stage 9 (Fig. 1C–C”’) and stage 10 (Fig. 1D–D”’). However, during the same period of oogenesis, ApVas1 was not colocalised with Osk in the posterior germ plasm (Fig. 1E–E”’”’). Instead, we could only visualise a weak expression of ApVas1 in the lateral cortex of the oocytes (Fig. 1E,F).

Bottom Line:
We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation.We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species.This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

ABSTRACTFormation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.